Recently, animal cell culture techniques have been remarkably developed, and also research and development related to animal cells have been carried out in a wide variety of fields. In dealing with animal cells of interest, not only the originally developed animal cells per se as well as the products thereof have been commercialized, but the cells or their cell surface proteins have come to be analyzed to design useful pharmaceuticals, regenerate patient's cells in vitro, or return them after enhancing the function to the patient's body for therapy. At present, the technology of culturing, as well as evaluating, analyzing and using the animal cells are a field that are attracting researchers' attention. Many of the animal cells including human cells are attachment-dependent. Thus, when animal cells are intended to be cultured in vitro, first they must be attached to the surface of a substrate. Sometimes it is necessary to detach the cultured cells, without separating into pieces, while retaining the form as they are cultured on the surface of the substrate.
In particular, with regard to the technology of regenerating the patient's cells in vitro, organ transplantation which intends to replace the incurable organ with another person's organ has become popular in recent years. Targets include a wide variety of organs such as the skin, the cornea, the kidney, the liver, and the heart, and prognosis after surgery has become extremely well, indicating that the technology is being established as one medical technology. Take corneal transplant as an example. Eye Bank was established in Japan about 50 years ago, along with the start of transplantation activities. However, the number of donors is still small, and while patients who require corneal transplant is about 20,000 in Japan alone, the actual number of patients who can receive the transplantation is said to be one tenth, or about 2000. Despite that corneal transplantation is an mostly established technology, a further improved medical technology is being sought after, due to shortage of donors. Under these circumstances, a technique of culturing the patient's normal cells to the desired size for transplantation was developed.
Under these circumstances, Japanese Unexamined Patent Publication (Kokai) No. 02-211865 (Patent Document 1) discloses a novel method of culturing cells on a cell culture support, where the surface of the substrate is coated with a polymer having an upper or lower critical solution temperature of 0 to 80° C. in water, the cells are cultured at a temperature not exceeding the upper critical solution temperature or not falling below the lower critical solution temperature, and the cultured cells are detached by increasing or decreasing the temperature of the substrate to exceed the upper critical solution temperature or fall below the lower critical solution temperature, without treatment with an enzyme. Japanese Unexamined Patent Publication (Kokai) No. 05-192138 (Patent Document 2) also describes a method of culturing skin cells using this temperature-responsive cell culture substrate at a temperature not exceeding the upper critical solution temperature or not failing below the lower critical solution temperature and then detaching the cultured skin cells with low damage by increasing or decreasing the temperature of the substrate to exceed the upper critical solution temperature or fall below the lower critical solution temperature. Use of the temperature-responsive cell culture substrate has led to a variety of new developments on known, culture techniques. Furthermore, in Japanese Unexamined Patent Re-publication (Saikohyo) No. 02-008387 (Patent Document 3), it was found that by culturing myocardial cells on a cell culture support the surface of which being coated with a temperature-responsive polymer, obtaining a myocardial cell-like sheet, then allowing the cultured layered cell sheet to adhere to a polymer membrane at a medium temperature not exceeding the upper critical solution temperature or not falling below the lower critical solution temperature, detaching it as it is together with the polymer membrane, and changing it into a three dimensional structure by a given method, a cell sheet having few structural defects and several functions as a myocardium-like tissue in vitro and a three-dimensional structure can be obtained. However, With the conventional technology described above, the myocardium-like cell sheet cannot be infinitely layered, with about three layers being the limit, and thus there has been a strong need for a technology that easily permits layering for a plurality of times.
In order to resolve the above problems, FASEB, J., 20(6), 708-710 (2006) (Non-patent document 1) attempted to multilayer cell sheets in vivo, and obtained a multilayered myocardial sheet with a thickness of 1 mm. It was found that in order to obtain a thick multilayered cell sheet among them, nutrients and oxygen must be supplied to each multilayered cell and each cell sheet. However, in the method of FASEB. J., 20(6), 708-710 (2006) (Non-patent document 1), cell sheets must be repeatedly transplanted in vivo, and thus the implanted site must be opened each time, which poses a great burden to the recipient of the transplant. Thus, there has been a strong need for a technology that permits simple multilayering for a plurality of times.